Development of quality control parameters for the standardization of Limonia acidissima L. leaf and stem

ObjectiveTo evaluate the pharmacognostic characters of Limonia acidissima L. (L. acidissima) leaf and stem, an important traditional medicinal plant.MethodsThe present study provides pharmacognostic, physicochemical and phytochemical details of leaf and stem of L. acidissima. Micro and macroscopic characters were analyzed. WHO recommended parameters were followed in the entire study.ResultsThe macroscopic study showed that the leaf was alternate, imparipinnately compound leaf with entire margin, long petiole, apex obtuse and base decurrent, with surface appearance and txture glabrous. The inflorescence was lateral and terminal panicles. The microscopic study of leaf revealed the presence of actinocytic stomata, multicellular trichome, prismatic calcium oxalate crystals, vascular bundles, etc. The powder microscopy also revealed prism like calcium oxalate crystals, multicellular trichome and actinocytic stomata. Physiochemical analysis of dried leaf powder showed total ash, water soluble ash and acid insoluble ash as 9.33%, 1.83% and 1.16% w/w respectively. Preliminary phytochemical screening revealed the presence of maximum amount of flavonoids and tannins. The main microscopic characteristic of stem was 2-3 layers of phellem, phellogen 2-3 layered followed by 7-8 layered phelloderm. Among other microscopic components were presence of xylem parenchyma, xylem vessels, xylem fibres and tracheids. The powder microscopy also revealed presence of annular, spiral vessel, prism crystals and multicellular trichome. Physiochemical analysis of dried stem powder showed total ash, water soluble ash and acid insoluble ash as 3.16%, 0.66% and 0.66% w/w respectively. Preliminary phytochemical screening showed the presence of maximum amount of only flavonoids.ConclusionsVarious pharmacognostical characters observed in this study will help in botanical identification and standardization of leaf and stem of L. acidissima and will also help in quality control and formulation development.     

Electrochemical,spectroscopic, and molecular docking studies of the interaction between the anti-retroviral drug indinavir and dsDNA

In this study, an electrochemical DNA biosensor was developed using a straightforward methodology to investigate the interaction of indinavir with calf thymus double-stranded deoxyribonucleic acid (ct-dsDNA) for the first time. The decrease in the oxidation signals of deoxyguanosine (dGuo) and deoxyadenosine (dAdo), measured by differential pulse voltammetry, upon incubation with different concentrations of indinavir can be attributed to the binding mode of indinavir to ct-dsDNA. The currents of the dGuo and dAdo peaks decreased linearly with the concentration of indinavir in the range of 1.0–10.0 μg/mL. The limit of detection and limit of quantification for indinavir were 0.29 and 0.98 μg/mL, respectively, based on the dGuo signal, and 0.23 and 0.78 μg/mL, respectively, based on the dAdo signal. To gain further insights into the interaction mechanism between indinavir and ct-dsDNA, spectroscopic measurements and molecular docking simulations were performed. The binding constant (K_b) between indinavir and ct-dsDNA was calculated to be 1.64 × 10⁸ M⁻¹, based on spectrofluorometric measurements. The obtained results can offer insights into the inhibitory activity of indinavir, which could help to broaden its applications. That is, indinavir can be used to inhibit other mechanisms and/or hallmarks of viral diseases.     

Evaluation of a chromogenic commercial assay using VWF-73 peptide for ADAMTS13 activity measurement

Introduction

Thrombotic thrombocytopenic purpura (TTP) is a thrombotic microangiopathy (TMA), related to a severe functional deficiency of ADAMTS13 activity (< 10% of normal). ADAMTS13 activity is thus crucial to confirm the clinical suspicion of TTP, to distinguish it from other TMAs, and to perform the follow-up of TTP patients.

Material and methods

We compared the performance of the commercial chromogenic assay Technozym® ADAMTS13 Activity ELISA (chromogenic VWF73 substrate, Chr-VWF73, Technoclone, Vienna, Austria), to that of our in-house FRETS-VWF73 used as reference method. A large group of 247 subjects (30 healthy volunteers and 217 patients with miscellaneaous TMAs) was studied.

Results

The lower limit of detection of the Chr-VWF73 was 3%, which is well adapted to the clinically relevant threshold for TTP diagnosis (10%). Our results showed a reasonable agreement between FRETS-VWF73 and Chr-VWF73 assays to distinguish samples with an ADAMTS13 activity < 10% from those with an ADAMTS13 activity > 10%. However, Chr-VWF73 assay provided false negative results in ~ 12% of acute TTP patients. Inversely, the Chr-VWF73 assay globally underestimated ADAMTS13 activity in detectable values ranging from 11 to 100% (with a great variability compared to FRETS-VWF73), which may be a concern for the follow-up of TTP patients in remission.

Conclusion

In-house assays developed and performed by expert laboratories remain the reference methods that should be used without limitation to control values provided by commercial assays when needed. Also, the development of an international reference preparation will be crucial to improve standardization.     

乙型肝炎血清标志物和HBV-DNA载量相关性分析

目的 探讨乙肝血清标志物与病毒DNA水平之间的相关性.方法 采用实时荧光定量PCR对874例HBV感染者血清中HBV-DNA含量进行检测,同时运用ELISA法检测HBV血清标志物,并统计分析患者乙型肝炎血清标志物、病毒DNA之间的相关性及分布特点.结果 在受检的874例标本中,男性533例,阳性率58.16％ （310/533）;女性341例,阳性率50.44％（172/341）,男性和女性阳性率比较差异有统计学意义（X2=5.01,P＜0.05）,男性和女性HBV-DNA水平差异无统计学意义（t =0.117,P=0.907）.乙肝总阳性率和HBV-DNA水平均随年龄的增长呈下降趋势,不同年龄组间比较差异有统计学意义.同一年龄段的大三阳与小三阳、两头阳和三抗阳之间比较差异有统计学意义;其中男性和女性HBV-DNA水平随年龄增长均呈下降趋势,差异有统计学意义.≤20岁以下人群HBV-DNA阳性率最高达82.86％.结论 HBV-DNA阳性率和HBV-DNA水平都随年龄增长呈下降趋势,其中≤20岁年龄段HBV-DNA阳性率最高达82.86％;男性HBV-DNA的阳性率高于女性.     

Fluorescence microscopic visualization of non cellular components during initial bioadhesion in situ

ObjectiveThe formation of an intraoral biofilm is primarily determined by initial bioadhesion processes, including molecular interactions. Therefore, this study aimed to establish fluorescent labelling protocols to enable the simultaneous visualization of different pellicle enzymes, extracellular glucans and adherent bacteria throughout the initial phase of biofilm formation.DesignIn situ formed biofilm samples were collected on enamel and dentine slabs that were fixed on buccal sites of individual splints, being worn by 5 subjects. After an intraoral slab exposure from 30 min to 8 h, the following specially adapted fluorescent labelling assays were performed and analyzed by epifluorescent microscopy: pellicle-amylase, -lysozyme, -peroxidase and -glycosyltransferases B, C and D were marked with specific primary antibodies and then visualized by the aid of different fluorescently labelled secondary antibodies (Texas Red, DyLight 488, FITC). Afterwards the same samples were subjected to a combined DAPI-/Concanavalin A-staining to determine adherent bacteria and glucans.ResultsAll fluorescence labelling assays were successfully established to visualize pellicle enzymes, glucans and adherent bacteria at different times of biofilm formation. The combination of the labelling protocols showed a characteristic agglomeration of glucans and bacteria as well as an increased concentration of the pellicle enzymes in the initial phase of bioadhesion.ConclusionFluorescent labelling techniques are a valuable supplement of dental research as they provide an insight into the mutual interactions of different biofilm determinants in situ. Based hereon, information could also be deduced about the influence of oral therapeutics on individual caries susceptibility.     

Lutetium(III) acetate phthalocyanines for photodynamic therapy applications: Synthesis and photophysicochemical properties

BackgroundThe development of new water-soluble photosensitizers for photodynamic therapy (PDT) applications is a very active research topic. Efforts have been made to obtain the far-red absorbing phthalocyanine complexes with molecular design that facilitates the uptake and selectivity for a high PDT efficiency.MethodsThe monomolecular lutetium(III) acetate phthalocyanines (LuPcs) substituted with methylpyridyloxy groups at non-peripheral (5) and peripheral (6) positions were synthesized by following the modification of the well-known synthetical routes. The photo-physicochemical properties of the both quaternized LuPcs were evaluated by the steady-state and time-resolved spectroscopy. The photochemical technique was applied to study the generation of the singlet oxygen.ResultsTwo water-soluble and cationic LuPcs were synthesized and chemically characterized. The photo-physicochemical properties of absorption (675 and 685 nm) and the red shifted fluorescence (704 and 721 nm) as well as the fluorescence lifetimes (2.24 and 3.27 ns) were studied. The promising values of singlet oxygen quantum yields (0.32 for 5 and 0.35 for 6) were determined.ConclusionsLutetium(III) acetate phthalocyanine complexes were synthesized and evaluated with physicochemical properties suitable for future photodynamic therapy applications.     

Fluorescence guided resection (FGR): A primer for oncology

Curative treatment for most cancer patients requires surgical removal of the tumor. Often though, residual disease is left behind negatively impacting tumor control and survival. Florescent Guided resection (FGR) is one type of Image Guided Surgery that offers the potential to improve outcomes for these patients. Currently, during FGR, a probe is preoperatively applied and allowed to concentrate in the tumor bed. At surgery appropriate light is applied to generate fluorescence which allows the surgeon to better visualize tumor extent. This improved visualization has translated both into enhanced rates for resection but also diminished rates of morbidity as less normal tissue need be removed to achieve negative margins. This paper will summarize the theory and practise of FGR as currently applied in clinical oncology including select outcomes and limitations of technique and technology.     

Fluorescence in situ hybridization in diagnostic cytology

Fluorescence in situ hybridization (FISH) is a technique that uses fluorescently labeled DNA probes to detect chromosomal alterations in cells. FISH can detect various types of cytogenetic alterations including aneusomy (ie, abnormalities of chromosome copy number), duplication, amplification, deletion, and translocation. Because tumor cells generally contain chromosomal alterations, FISH is able to detect cells that have chromosomal abnormalities consistent with neoplasia in exfoliative and aspiration cytology specimens. This review will discuss the utility of FISH for the detection of bladder, lung, pancreatobiliary, and esophageal carcinoma in cytologic specimens.     

Fluorescence, birefringence and confocal microscopy of the abdominal aorta from nonobese diabetic (NOD) mice

In this study, nonenzymatic glycosylation was assessed in aorta extracellular matrix (ECM) from nonobese diabetic (NOD) mice, using nitroblue tetrazolium (NBT). Molecular and structural changes were investigated in elastic lamellae and collagen fibers of diabetic mice aortas after staining with dansyl chloride and anilinonaphthalene sulfonate (ANS). Alterations in arterial autofluorescence and birefringence of collagen fibers were investigated in unstained aortas. Proliferation of smooth muscle cells (SMC) was also investigated by Feulgen reaction staining assessed by confocal microscopy and image analysis. Assessment of nonenzymatic glycosylation demonstrated glycosylation products in the aorta ECM of NOD mice. Elastic lamellae and collagen fibers from NOD mouse aortas presented less intense fluorescence after staining with dansyl chloride and ANS when compared to aortas of control nondiabetic mice. However, unstained NOD aortas showed more intense autofluorescence when compared to controls. Birefringence analysis suggests alterations in the higher molecular packing of the arterial collagen fibers in NOD aortas. In aortas stained by Feulgen reaction, no evidence of SMC proliferation was observed in diabetic aortas.